SUMMARY Biofilms in the human middle-ear are known to be directly associated with chronic otitis media (OM), and the bacteria that reside within these protective biofilms are likely responsible for contributing to the persistence and re-seeding of the infection despite repeated doses of antibiotics. While prior sampling of human middle-ear biofilms was performed invasively during a surgical procedure, developments and advances in the use of innovative optical imaging technologies and systems have made it possible to non-invasively detect, characterize, and monitor any biofilm that may be affixed to the tympanic membrane and/or any effusion that may be present within the middle ear. Optical coherence tomography (OCT) has been developed for imaging applications in otolaryngology and primary care, and over the last four years, feasibility studies have demonstrated its use for imaging various tissue sites examined during a primary care exam. In this renewal project, we propose to fundamentally investigate both the clinical and biological significance of the middle-ear biofilms and effusions that are detectable with OCT systems, as well as develop low-cost solutions that will enable this technology to be economical for primary care physicians and front-line healthcare providers. Guided by a central hypothesis and five specific aims, we intend to successfully demonstrate how non-invasive OCT of the middle ear can be used to improve the clinical management of OM by identifying the presence and dynamics of biofilms and effusions in this highly prevalent disease. To accomplish our goals, we have evolved and expanded our successful bioengineering research partnership to include new world-renowned expertise in the biological and clinical investigation of middle ear biofilms, as well as a start-up company that emerged during our initial project period to fully translate and commercialize this novel technology. We expect that this new technology and its capabilities will provide new metrics for improved detection, diagnosis, and monitoring of OM. The long-term outcome and impact will be a new standard-of-care for the management of ear infections, with improved patient care.